CN112933239A - Application of reagent for activating endogenous PD-1 of tumor cells in preparation of antitumor drugs - Google Patents

Application of reagent for activating endogenous PD-1 of tumor cells in preparation of antitumor drugs Download PDF

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CN112933239A
CN112933239A CN202110164143.6A CN202110164143A CN112933239A CN 112933239 A CN112933239 A CN 112933239A CN 202110164143 A CN202110164143 A CN 202110164143A CN 112933239 A CN112933239 A CN 112933239A
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cancer
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tumor cells
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王冬来
曹治杰
刘雅菁
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Institute of Basic Medical Sciences of CAMS
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract

The invention relates to application of a reagent for activating endogenous PD-1 of tumor cells in preparation of an anti-tumor medicament. In particular, the present invention relates to the use of a combination of a histone deacetylase inhibitor and an anti-tumour agent that activates the p53 pathway in the preparation of a medicament for treating a tumour in a subject. In another aspect, the invention relates to a method for enhancing the expression of endogenous PD-1 in a tumor cell in vitro, comprising: 1) culturing tumor cells in a constant-temperature cell culture box at 37 ℃ for 1 day; 2) adding an antitumor agent which activates the p53 pathway to the tumor cells of step 1), and culturing the tumor cells for 18 hours; and 3) adding the trichostatin A and the nicotinamide to the tumor cells obtained in the step 2) to treat the cells for 6 hours.

Description

Application of reagent for activating endogenous PD-1 of tumor cells in preparation of antitumor drugs
Technical Field
The invention relates to the technical field of medicinal chemistry, and particularly provides a combined administration mode of key molecules of targeted tumor cells. According to the method disclosed by the invention, the endogenous PD-1 expression of the tumor cells can be obviously improved.
Background
Recent global cancer data shows that in the tens of millions of cancer deaths worldwide, cancer has been the first killer of human life. China always puts the life safety and body health of the people at the first place and attaches importance to the development of science and technology and medical treatment. With the development of science and technology and medical health, cancer treatment has not been limited to general surgery, radiotherapy, chemotherapy or targeted therapy.
In recent years, the monoclonal antibody immunotherapy of an immune checkpoint inhibitor PD-1/PD-L1 has clinically significant curative effects. In nearly 20 solid tumors including advanced melanoma, lung cancer, breast cancer, skin cancer, lymphoma and the like, the PD-1/PD-L1 monoclonal antibody helps the T cells in immune cells to restore the activity by blocking the interaction between PD-1 receptors on the surface of the immune cells and PD-L1 ligands on the surface of tumor cells, thereby killing cancer cells and substantially improving the survival period of patients with advanced tumors. However, as clinical treatment cases increase, the difference between patients is large, and drug resistance, side effects or no response become obvious gradually, which becomes a great obstacle to immunotherapy, and therefore, a therapeutic regimen based on PD-1 as a target needs to be further improved.
Recent studies have found that PD-1 is also expressed in tumor cells and regulates the biological behavior of tumor cells in an immune independent manner. In melanoma cells, liver cancer cells, pancreatic cancer cells and lung cancer cells, the endogenous PD-1 of the tumor cells is found to be involved in regulating and controlling the proliferation of the tumor cells. Because the expression level of the endogenous PD-1 of the tumor cells is extremely low, a method for activating the endogenous PD-1 of the tumor cells needs to be developed, and a new medication strategy is provided for a treatment method based on the tumor cells PD-1.
In addition, studies have found that Histone Deacetylase (HDAC) levels are markedly abnormal in some cancers; HDACs can remove histone acetyl groups to make chromosomes more compact, thereby inhibiting transcription of genes. Therefore, HDAC inhibitors (HDACi) can promote gene expression by increasing acetylation levels to achieve the effect of treating cancer. In 2006, the FDA approved HDACi, vorinostat, developed by mercker for the treatment of cutaneous T-lymphomas. However, HDACI has poor selectivity, so that the HDACI is frequently combined with other tumor drugs to be applied to tumor treatment.
At present, there is still a need to develop a combined drug capable of effectively treating tumors, which can both target tumors and effectively enhance the effectiveness of current antitumor drugs.
Disclosure of Invention
The present invention is based on the following unexpected findings: Nutlin-3A or Camptothecin (CPT), trichothecin A (TSA) and Nicotinamide (NAM) can obviously increase the expression of endogenous PD-1 of tumor cells and effectively inhibit the growth of the tumor cells after the tumor cells are treated in vitro.
Nutlin-3A is an active isomer of Nutlin-3, is a murine double minute 2(MDM2) antagonist, inhibits MDM2-p53 interaction, and stabilizes p53 protein, thereby inducing cell cycle arrest and apoptosis.
Camptothecin (CPT), an alkaloid extracted from seed or root bark of camptotheca acuminata of davidiaceae, can directly damage DNA structure, bind to DNA to make DNA vulnerable to endonuclease, inhibit DNA polymerase to affect DNA replication, is mainly sensitive to proliferating cells, is a cell cycle specific drug, acts on S phase, and has slight lethality to G1, G2 and M phase cells.
Trichostatin a (TSA) is a histone deacetylase inhibitor (HDACi).
Nicotinamide (NAM), also known as Nicotinamide, is an amide compound of nicotinic acid. A crystalline powder that is white; no or almost no odor, bitter taste; it is slightly hygroscopic. It is easily soluble in water or ethanol, and soluble in glycerol. The traditional Chinese medicine composition is mainly used for preventing and treating pellagra, stomatitis, glossitis, sick sinus syndrome, atrioventricular block and the like in clinic. In addition, nicotinamide, an inhibitor of histone deacetylase Sir2 α, inhibits Sir2 α -mediated deacetylation of p53, rendering p53 acetylated, and plays an important role in the performance of p53 functions (see Luo, J., et al, 2001.'Negative control of p53 by Sir2alpha proteins Cell subset stress', Cell,107: 137-48).
The present inventors have unexpectedly found that endogenous PD-1 in tumor cells is involved in the p 53-dependent tumor suppressor pathway, based on which the present inventors propose the following scheme.
Thus, in a first aspect, the present invention provides the use of a combination of a histone deacetylase inhibitor (HDACi) and an anti-tumour agent that activates the p53 pathway in the preparation of a medicament for the treatment of a tumour, preferably, which inhibits tumour growth in a subject.
In preferred embodiments, the histone deacetylase inhibitors include, but are not limited to: trichostatin a (Trichostatin a, TSA), nicotinamide, trapoxin B, phenylbutyrate (phenylbutyrate), valproic acid, vorinostat (suberaninolamic acid or SAHA, as a commercial product), belinostat (belinostat, PXD101, as a commercial product), panobinostat (panobinostat, as a commercial product), dacinostat (dacinostat, LAQ824), entinostat (entinostat, SNDX-275 or MS-275), tacalan (tacedinaline, CI994) and moxistat (MGCD 0103).
Further preferably, said antineoplastic agent which activates the p53 pathway is an MDM2-p53 inhibitor or a cytotoxic agent, e.g., Nutlin-3A, or camptothecin.
The tumor is non-small cell lung cancer, osteosarcoma, breast cancer, colorectal cancer, gastric cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostatic cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma or/and cholangiocarcinoma; preferably non-small cell lung cancer or osteosarcoma.
Preferably, the subject according to the invention is a mammalian subject, preferably the subject is a human.
A method for increasing the expression of tumor cell endogenous PD-1 by the combined administration of key molecules of targeted tumor cells.
In another aspect, the invention provides a method for enhancing in vitro expression of endogenous PD-1 in a tumor cell, the method comprising the following:
1) culturing tumor cells in a constant-temperature cell culture box at 37 ℃ for 1 day;
2) adding an antitumor agent which activates the p53 pathway to the tumor cells of step 1), and culturing the tumor cells for 18 hours; and
3) adding trichostatin A and nicotinamide into the tumor cells obtained in the step 2), and treating the cells for 6 hours.
In a preferred embodiment, wherein the tumor cell is a p53-Tet-on inducible p53 stably expressed H1299 cell line, the antineoplastic agent that activates the p53 pathway in step 2) is doxycycline; when the tumor cell is osteosarcoma cell U2OS, the antitumor agent activating p53 pathway in step 2) is selected from camptothecin or Nutlin-3A.
Preferably, the doxycycline in the step 2) is dissolved in water, and the concentration of the working solution is 1 mug/mL; camptothecin or Nutlin-3A was dissolved in DMSO/diluted with water, and the concentration of the working solution of camptothecin was 0.1-1. mu.M, and the concentration of the working solution of Nutlin-3A was 10. mu.M.
Preferably, the concentration of the working solution in the step 3) is 1 mu M, wherein the concentration of the trichostatin A is DMSO dissolution/water dilution; nicotinamide was dissolved in water at a working solution concentration of 5 mM.
Drawings
Fig. 1A and 1B: the doxycycline and HDACI combined significantly enhance the expression of endogenous PD-1 in non-small cell lung cancer cells. FIG. 1A: detecting the mRNA expression level of PD-1 in H1299p53-Tet-on cells by an RT-qPCR technology after 24 hours of doxycycline (Doxy) treatment and/or the last 6 hours of TSA + NAM treatment; FIG. 1B: protein expression levels of PD-1 in H1299p53-Tet-on cells were examined by Western blotting technique at 24 hours of doxycycline (Doxy) treatment and/or at the last 6 hours of TSA + NAM treatment.
Fig. 2A to 2D: CPT or Nutlin-3A combined with HDACI significantly enhanced the expression of endogenous PD-1 in osteosarcoma U2OS cells. FIG. 2A: changes in mRNA levels of PD-1 in CPT alone and in combination with TSA + NAM treated U2OS cells; FIG. 2B: changes in protein levels of PD-1 in CPT alone and in combination TSA + NAM treated U2OS cells; FIG. 2C: changes in mRNA levels of PD-1 in U2OS cells treated with Nutlin-3A alone and in combination with TSA + NAM; FIG. 2D: changes in protein levels (D) of PD-1 in U2OS cells treated with Nutlin-3A alone and in combination with TSA + NAM.
Fig. 3A to 3C: PD-1 in tumor cells regulates tumor cell growth in an immune independent manner. FIG. 3A: growth of mice and tumors after 30 days of mouse tumor-bearing experiments carried out by H1299-EV and H1299-PD-1 stable cell lines; FIG. 3B trend of tumor volume change of tumor-bearing experiment (FIG. 3A); FIG. 3C: tumor weight of tumor-bearing experiment (FIG. 3A).
Fig. 4A to 4C: tumor cell endogenous PD-1 is involved in p53 dependent tumor suppression. FIG. 4A: detecting the knocking-down efficiency of PD-1 in a H1299p53-Tet-on cell line by Western blotting; FIG. 4B: cell proliferation experiment analysis H1299p53-Tet-on sh-Ctr and sh-PD-1 cells grow under the action of Doxy and/or TSA + NAM drugs; FIG. 4C: quantitative analysis was performed based on the results of fig. 4B.
Detailed Description
To illustrate the present invention in more detail, the following specific embodiments are provided in the present specification and described with reference to the accompanying drawings, but the present invention is not limited thereto. The method and the use of the present invention can be appropriately modified by those skilled in the art in combination with the common general knowledge in the art, and the method and the use of the present invention are within the scope of the present invention as long as the functions of the present invention can be achieved.
The experimental methods, in which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions or according to conditions recommended by the supplier of the reagents, cells or kits. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.
Example 1: doxycycline combined HDACI (hdrocycline-associated antigen) can significantly enhance expression of endogenous PD-1 in non-small cell lung cancer cells
A p53-Tet-on inducible expression stable cell line (as described in Jiang, L.et al, 2015.Ferroptosis as a p53-mediated activity heavy tumor repair', Nature,520: 57-62) was constructed using a p53 deficient non-small cell lung cancer H1299 cell line (purchased from the national laboratory cell resource sharing service platform, resource number: 3111C0001CCC 000469). Inoculating the stable cell line into a 6-well plate, culturing in a constant-temperature cell culture box at 37 ℃, and adding a medicament for treatment after one day, wherein the non-medicament added is used as a negative control.
The specific implementation steps are as follows: the p53-Tet-on stable transfected cell line was inoculated into 6-well plates, cultured in DMEM puromycin resistant medium (containing 2. mu.g/mL puromycin, 100U/mL penicillin, 100. mu.g/mL streptomycin and 10% FBS), and placed in a cell culture box for culture. The next day, doxycycline 1 μ g/mL was added for 24 hr, HDACI drugs (1 μ M TSA and 5 μ M NAM) were added for 18hrs in the combined treatment group, and then for 6 hr, and doxycycline and HDACI drug-independent treatment groups were set. Finally, cells are harvested for mRNA and protein level measurements.
The experimental results show that doxycycline treatment group alone induces p53 expression, and simultaneously detects the increase of endogenous PD-1mRNA (figure 1A) and protein expression level (figure 1B) in lung cancer cells; the expression of PD-1 is obviously increased in a combined treatment group of doxycycline and HDACI medicines; whereas HDACi treated the group alone with only a slight increase. Thus, the administration of the tumor cell targeting p53 molecule in combination with HDACi can activate the expression of endogenous PD-1 in lung cancer cells.
Example 2: CPT or Nutlin-3A combined with HDACI remarkably enhances the expression of endogenous PD-1 in osteosarcoma U2OS cells
The specific implementation steps are as follows: osteosarcoma cells U2OS (purchased from national laboratory cell resource sharing service platform, resource number: 3111C0001CCC000028) were inoculated into 6-well plates, cultured in DMEM medium (containing 10% FBS), and placed in a cell culture box for culture. The next day, CPT 1. mu.M or Nutlin-3A 10. mu.M was added for 24 hours, the combination treatment group was treated with HDACI drugs (1. mu.M TSA and 5. mu.M NAM) for 18 hours and then treated for 6 hours with CPT or Nutlin-3A, and a CPT/Nutlin-3A, HDACi drug-only treatment group was set. Finally, cells are harvested for mRNA and protein level measurements.
Experimental results see figures 2A to 2D, which show that CPT or Nutlin-3A alone treatment group can target increased p53 expression, while increased expression of endogenous PD-1mRNA and protein levels in osteosarcoma U2OS cells was detected; in the combined treatment group of CPT or Nutlin-3A and HDACI medicaments, the expression of PD-1 is increased more obviously; whereas in the HDACi drug-treated group alone, there was only a slight increase in PD-1 expression. Thus, it was shown that the administration of a molecule targeting tumor cell p53 in combination with HDACi also enhanced the expression of endogenous PD-1 in osteosarcoma U2OS cells.
Example 3: PD-1 in tumor cells regulates tumor cell growth in an immune-independent manner
The method comprises the following specific steps:
(1) the p53 deficient H1299 Cell line is used for constructing a PD-1 overexpression stable Cell line, and the specific steps are described in Chu, B. et al, 2019.ALOX12 is required for p53-mediated tissue repair surgery pathway, Nat Cell Biol,21: 579-91;
(2) will 3 x 106Respectively mixing PD-1 over-expressed or control cells and substrate glue uniformly in a ratio of 1.5:1, injecting 200 mu L of cell substrate glue mixed liquor to the subcutaneous part of an immune full-defect 6-week-old B-NDG mouse, inoculating the control cells on the left side of each mouse, and inoculating the PD-1 over-expressed cells on the right side of each mouse;
(3) counting the length and width of the tumor after one week according to the formula: length (width)2Dividing by 2, calculating the tumor volume, and counting every 2-3 days;
(4) the experiment was terminated when the tumor-bearing experiment was carried out for 30 days, and subcutaneous tumors of mice were collected for photographing, weighing and data analysis.
The results of the experiments (as shown in FIGS. 3A to 3C) show that the expression of PD-1 in tumor-bearing experiments in immunocompromised mice, both in terms of the weight of the tumor and in terms of the growth tendency of the tumor, exerts an inhibitory effect on the growth of tumor cells, and that this inhibitory effect is an immune-independent form.
Example 4: tumor endogenous PD-1 is involved in p53 dependent tumor suppression
The method comprises the following specific steps:
(1) in the H1299p53-Tet-on stable cell line, the cell lines of H1299p53-Tet-on sh-PD-1 and sh-Ctr are selected by using lentivirus to interfere the expression of PD-1, as shown in Jiang, L., et al 2015.Ferroptosis as a p53-mediated activity heavy task study supply, Nature 520: 57-62.
(2) H1299p53-Tet-on sh-PD-1 and sh-Ctr cells are inoculated into a 6-well plate, a non-drug-added group is designed as a negative control, treatment groups are respectively Doxy, TSA + NAM and Doxy combined TSA + NAM treatment, and each group has 3 repeats.
(3) After 2 days of drug-added culture, the mixture was fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then stained with 0.1% crystal violet and photographed.
(4) The crystal violet stained cells were lysed with 10% acetic acid for 30 minutes, and the absorbance was measured by a microplate reader to give relative counts and statistical analysis to each set of cells.
The results of the experiments are shown in FIGS. 4A to 4C, which show that p53 expression inhibits tumor cell growth when Doxy is administered alone; when TSA + NAM is singly administered, the inhibition effect on the growth of tumor cells is weak; and when Doxy and TSA + NAM are jointly administered, namely p53 and PD-1 are simultaneously activated in the tumor cells, the growth of the tumor cells is remarkably inhibited. Knockdown of PD-1 decreased the inhibitory effect of the drug on tumor cell growth compared to control cells. This suggests that tumor cell endogenous PD-1 is involved in p 53-dependent tumor suppression.

Claims (10)

1. Use of a histone deacetylase inhibitor in combination with an anti-tumour agent that activates the p53 pathway in the manufacture of a medicament for treating a tumour in a subject.
2. The use according to claim 1, wherein the histone deacetylase inhibitor is selected from one or more of the group consisting of trichostatin A, nicotinamide, trapoxin B, phenylbutyrate, valproic acid, vorinostat, belinostat, panobinostat, dacisistat, entinostat, tadalafil and moxystat, preferably is trichostatin A and nicotinamide.
3. The use according to claim 1 or 2, wherein the antineoplastic agent which activates the p53 pathway is selected from Nutlin-3A or camptothecin.
4. The use of any one of claims 1-3, wherein the tumor is selected from non-small cell lung cancer, osteosarcoma, breast cancer, colorectal cancer, gastric cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma, or/and cholangiocarcinoma; preferably non-small cell lung cancer or osteosarcoma.
5. Use of a combination of an anti-tumour agent that activates the p53 pathway, trichostatin A and nicotinamide in the manufacture of a medicament for treating a tumour in a subject.
6. The use of any one of claims 1-5, wherein the medicament inhibits tumor growth in a subject.
7. The use of any one of claims 1-6, wherein the subject is a human.
8. A method for enhancing in vitro endogenous PD-1 expression in a tumor cell, the method comprising the steps of:
1) culturing tumor cells in a constant-temperature cell culture box at 37 ℃ for 1 day;
2) adding an antitumor agent which activates the p53 pathway to the tumor cells of step 1), and culturing the tumor cells for 18 hours; and
3) adding the trichostatin A and the nicotinamide to the tumor cells of the step 2) to treat the cells for 6 hours.
9. The method of claim 8, wherein when the tumor cell is a H1299 cell line that can induce stable expression of p53 by the p53-Tet-on system, the anti-tumor agent that activates the p53 pathway in step 2) is doxycycline; when the tumor cell is osteosarcoma cell U2OS, the antitumor agent activating p53 pathway in step 2) is selected from camptothecin or Nutlin-3A.
10. The method according to claim 9, wherein the doxycycline in step 2) is dissolved in water, and the working solution concentration is 1 μ g/mL; dissolving camptothecin or Nutlin-3A in DMSO/diluting with water, wherein the concentration of the working solution of camptothecin is 0.1-1 μ M, and the concentration of the working solution of Nutlin-3A is 10 μ M;
in the step 3), the concentration of the working solution is 1 mu M, wherein the trichostatin A is dissolved in DMSO/diluted with water; nicotinamide was dissolved in water at a working solution concentration of 5 mM.
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CN115975942A (en) * 2023-02-27 2023-04-18 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) Pancreatic cancer immunotherapy drug-resistant cell line and preparation method and application thereof
CN115975942B (en) * 2023-02-27 2023-09-22 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) Pancreatic cancer immunotherapy drug-resistant cell line, and preparation method and application thereof

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